PROJECT SUMMARY The purpose of this five-year proposal is to provide an integrative and personalized training program for the applicant to transition into an independent academic position as a basic scientist focused on understanding the underlying causes of congenital heart disease. The career development plan will provide additional training in induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) phenotyping, CRISPR/Cas9 genome editing, and bioinformatics. The applicant will also receive a wealth of informal and didactic training at Stanford University in specialized areas such as professional development and grant writing skills, which will be critical for the applicant to gain autonomy and launch a productive career as an independent investigator. Under the expert mentorship of Dr. Sean Wu and Dr. Euan Ashley, along with the assembled advisory committee (Dr. Daniel Bernstein, Dr. Marlene Rabinovitch, Dr. James Priest, and Dr. Matthew Porteus), the applicant will receive the necessary guidance and resources to accomplish these goals and efficiently transition to independence following the K08 training period. The research topic of this proposal fulfills a significant knowledge gap in the field by identifying myocyte- intrinsic contributions to single ventricle congenital heart disease. By using patient-specific iPSCs, intrinsic abnormalities of early cardiac development that contribute to human congenital heart disease can be studied. Among the most severe and lethal forms of congenital heart disease are single ventricle diseases, which involve profound underdevelopment of either the left or right ventricle, with resulting insufficient systemic or pulmonary blood flow. While the prevailing theory of disease pathogenesis involves impaired ventricular growth due to reduced blood flow in the developing ventricle, myocyte-intrinsic abnormalities are also suspected but largely unexplored. In Aim 1, intrinsic deficiencies in ventricular cardiomyocyte generation and function will be assessed. By using an elegant reporter system, specific abnormalities will be characterized in a chamber- specific manner and attributed to left or right ventricular iPSC-CMs. In Aim 2, the mechanisms behind myocyte- intrinsic perturbations will be investigated using single cell RNA sequencing analysis to identify abnormal gene expression patterns. Additionally, this data will be compared to ventricular tissue RNA sequencing data from the Pediatric Cardiac Genomics Consortium (PCGC) to validate the ability of iPSC-CMs to reflect diseased myocardium. Finally, in Aim 3 iPSC-CMs will be exposed to glucose and oxygen deprivation to mimic the effects of impaired blood flow during development, followed by an assessment of cell death and proliferation.